A device for separately modulating the wave fronts of two components of a collimated light beam, the components being transversally polarized in orthogonal polarization directions, is known from Lenz, Martin O. et al. “3-D stimulated emission depletion microscopy with programmable aberration correction”, J. Biophotonics 7, No. 1-2, 29-36 (2014). Here, in an STED microscope, fluorescence inhibiting light is provided as a collimated light beam comprising two components of transversal polarization directions which are orthogonal to each other. The wave fronts of the two components are one after the other modulated by a spatial light modulator (SLM) used in an off-axis holography configuration, the diffracted light being separated from the zero order reflection of the surface of the spatial light modulator. At first the fluorescence inhibiting light is directed onto a first partial area of a spatial light modulator to modulate the wave fronts of the horizontally polarized first component in such a way that a donut-shaped intensity distribution of the fluorescence inhibiting light results around the focus of a following objective. The vertically polarized polarization second component remains unchanged. Both components are then rotated by 90° by double passing the fluorescence inhibiting light through a quarter wave plate oriented at 45° in an image relay arm comprising the quarter wave plate, a lens and a mirror, such that the first partial area of the spatial light modulator is imaged onto a second partial area of the spatial light modulator. By being diffracted in this second partial area, only the now horizontally polarized second component of the fluorescence inhibiting light is modulated. Particularly, it is modulated in such a way that intensity maxima of the fluorescence inhibiting light are formed which, in the direction of the optical axis of an objective focusing the fluorescence inhibiting light, are arranged both in front of and behind the focus of the objective. Together with the donut-shaped intensity distribution of the first component of the fluorescence inhibiting light, the zero point of the fluorescence inhibiting light at the focus point of the objective is thus delimited in all spatial directions. The image relay arm also ensures that both components of the fluorescence inhibiting light will be collinear despite the diffraction they experience. Prior to focusing the two components of the fluorescence inhibiting light, they are both circularly polarized by means of a further quarter wave plate. Nevertheless, they do not interfere because they have been delayed with regard to each other for more than a coherence length of the fluorescence inhibiting light by means of a light guide fiber which delays one of the two components of the first and second polarization directions with regard to the other of the two components.
The device described by Lenz et al. is not easily integrated in an existing laser scanning microscope as its dimensions are much bigger than a focus length of optics imaging the spatial light modulator. Further, the mirror is arranged in the focus point of the lens of the image relay arm so that the focused light beam impinges on the mirror in one spot only. With a high power of the fluorescence inhibiting light, this results in very high light intensities which may damage the mirror. Further, the function of the known device is very sensitive to any contaminations of the mirror in the spot of incidence of the focused light beam.
DE 10 2007 025 688 A1 discloses an optical set-up comprising an objective for projecting two optically different light components into a common projection space and an optical part which deforms passing wave fronts of the one light component such that the intensity distribution of the one light component due to interference with itself differs from the intensity distribution of the other light component in the projection space. Both the wave fronts of the other light component and the wave fronts of the one light component pass the optical part which, however, does not deform the optical wave fronts of the other light component or which may at least be phase-corrected for the other light component. The two light components may differ in their polarization. Then, the optical part has birefringent optical properties. Particularly, the optical part may be a spatial light modulator by which the form of wave fronts of an axially polarized light component may be designed to a far extent whereas it leaves the wave fronts of light of other polarization directions unchanged.
WO 2010/133678 A1 discloses a laser scanning microscope with a birefringent chromatic device for beam forming. The microscope has a light source for excitation light and fluorescence inhibiting light, the excitation light and the fluorescence inhibiting light being components of a collimated light beam differing in wavelength. The birefringent chromatic device modulates the polarization distribution over the cross-section of the light beam differently for the excitation light and the fluorescence inhibiting light such that the excitation light comprises an intensity maximum at the focus of a following objective, whereas the fluorescence inhibiting light comprises a zero point at the focus of the objective which is surrounded by intensity maxima of the fluorescence inhibiting light.
From Muro, Mikio and Takatani, Yoshiaki “Optical rotatory-dispersion-type spatial light modulator and characteristics of the modulated light”, Applied Optics, Vol. 44, No. 19, 3992-3999 it is known to place a chromatic optical polarization rotator in front of a spatial light modulator operated in transmission. The polarization rotator selectively rotates one component of a linearly polarized light beam comprising two components of different wavelengths such that this first component has the first polarization direction for which the spatial light modulator is active. Thus, in an STED microscope, the fluorescence inhibiting light may selectively be modulated with regard to its wave fronts to provide a zero point at the focus of the objective which is, for example, surrounded by a ring-shaped intensity distribution of the fluorescence inhibiting light, whereas the excitation light passes the spatial light modulator without modulation of its wave fronts and is thus focused by the objective such that it has its intensity maximum at the focus point. The known device is only suited for forming an intensity distribution of the fluorescence inhibiting light which delimits the zero point of the intensity distribution of the fluorescence inhibiting light in two spatial dimensions but which does not delimit the zero point also in the third spatial direction, i.e. along the z-axis.
There still is a need of a device for separately modulating the wave fronts of two components of a collimated light beam, which have orthogonal transversal polarization directions, the device being so compact that it may be integrated in existing laser scanning microscopes to form, at a high operational reliability, an intensity distribution of fluorescence inhibiting light delimiting a zero point in the focus of an objective of the laser scanning microscope in all three spatial dimensions with intensity maxima of the fluorescence inhibiting light.